The Journal of Neuroscience, September 1, 1998, 18(17):7008–7014

Nitric Oxide Signaling in Pain and Nociceptor Sensitization in the Rat

K. O. Aley, Gordon McCarter, and Jon D. Levine Departments of Anatomy, Medicine, and Oral Surgery, Division of Neuroscience, and National Institutes of Health Pain Center (UCSF), University of California at San Francisco, San Francisco, California 94143-0452

We investigated the role of nitric oxide (NO) in inflammatory We next performed experiments to test whether administra- hyperalgesia. Coinjection of prostaglandin E2 (PGE2 ) with the tion of exogenous NO precursor or donor could mimic the G nitric oxide synthase (NOS) inhibitor N -methyl-L-arginine (L- hyperalgesic effect of endogenous NO. Intradermal injection of

NMA) inhibited PGE2-induced hyperalgesia. L-NMA was also either the NOS substrate L-arginine or the NO donor 3-(4- able to reverse that hyperalgesia. This suggests that NO con- morphinolinyl)-sydnonimine hydrochloride (SIN-1) produced tributes to the maintenance of, as well as to the induction of, hyperalgesia. However, this hyperalgesia differed from PGE2- PGE2-induced hyperalgesia. Consistent with the hypothesis induced hyperalgesia, because it was independent of the cAMP that the NO that contributes to PGE2-induced sensitization of second messenger system and blocked by the guanylyl cyclase primary afferents is generated in the dorsal root ganglion (DRG) inhibitor ODQ. Therefore, although exogenous NO induces hy- neurons themselves, L-NMA also inhibited the PGE2-induced peralgesia, it acts by a mechanism different from that by which increase in tetrodotoxin-resistant sodium current in patch- endogenous NO facilitates PGE2-induced hyperalgesia. Con- clamp electrophysiological studies of small diameter DRG neu- sistent with the hypothesis that these mechanisms are distinct, rons in vitro. Although NO, the product of NOS, often activates we found that inhibition of PGE2-induced hyperalgesia caused guanylyl cyclase, we found that PGE2-induced hyperalgesia by L-NMA could be reversed by a low dose of the NO donor was not inhibited by coinjection of 1H-[1,2,4]oxadiazolo[4,3- SIN-1. The following facts suggest that this dose of SIN-1 a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. We then mimics a permissive effect of basal levels of NO with regard to tested whether the effect of NO depended on interaction with PGE2-induced hyperalgesia: (1) this dose of SIN-1 does not the adenylyl cyclase–protein kinase A (PKA) pathway, which is produce hyperalgesia when administered alone, and (2) the known to mediate PGE2-induced hyperalgesia. L-NMA inhibited effect was not blocked by ODQ. hyperalgesia produced by 8-bromo-cAMP (a stable membrane In conclusion, we have shown that low levels of NO facilitate permeable analog of cAMP) or by forskolin (an adenylyl cyclase cAMP-dependent PGE2-induced hyperalgesia, whereas higher activator). However, L-NMA did not inhibit hyperalgesia pro- levels of NO produce a cGMP-dependent hyperalgesia. duced by injection of the catalytic subunit of PKA. Therefore, the contribution of NO to PGE2-induced hyperalgesia may Key words: hyperalgesia; nitric oxide; pain; primary afferent occur in the cAMP second messenger pathway at a point nociceptor; prostaglandin E2; protein kinase A; tetrodotoxin- before the action of PKA. resistant sodium current

Tissue injury results in hyperalgesic pain (tenderness), probably directly on the peripheral terminals of primary afferent nocicep- the most common presenting clinical symptom. This important tors to produce hyperalgesia (Taiwo and Levine, 1989), to sensi- phenomenon is believed to be attributable, in great part, to tize nociceptors in vitro (England et al., 1996; Gold et al., 1996a), sensitization of primary afferent nociceptors so that they respond and to enhance tetrodotoxin-resistant voltage-gated sodium cur- at a lower stimulus intensity and with greater number of action rent (TTX-R INa ). Previous work in our laboratory suggests that potentials. Inflammatory mediators have been implicated in pro- PGE2-induced sensitization of nociceptors is mediated by the ducing this sensitization and hyperalgesia. Of these, prostaglan- adenylyl cyclase–cAMP–protein kinase A (PKA) second mes- dins are well established as mediators of mechanical hyperalgesia senger system. For example, agents that inhibit adenylyl cyclase, in both animals and humans (Collier and Schneider, 1972; as well as those that inhibit PKA, attenuate PGE2-induced hy- Moncada et al., 1975; Ferreira et al., 1978) and of sensitization of peralgesia (Taiwo and Levine, 1989, 1991; Khasar et al., 1995). primary afferent nociceptors (Martin et al., 1987; Schaible and PGE2 is a key mediator of inflammatory hyperalgesia. Schmidt, 1988; Davis et al., 1993; Rueff and Dray, 1993). The A number of observations suggest that in the periphery nitric inflammatory mediator prostaglandin E2 (PGE2 ) is thought to act oxide (NO) also acts as a pronociceptive mediator (Moulton, 1996; Robbins and Grisham, 1997; Wallace and Chin, 1997). Received April 23, 1998; revised June 12, 1998; accepted June 17, 1998. Intracutaneous injections of NO precursors evoke pain in humans This work was supported by National Institutes of Health Grant NS21647. We thank Dr. David Bredt for many discussions about NO/cGMP signaling and Drs. (Houlthusen and Arndt, 1994, 1995). That NO is generated David Reichling and Kimberly Tanner for their careful scrutiny of this manuscript. within nociceptors is suggested by these observations: (1) neuro- Correspondence should be addressed to Dr. Jon D. Levine, National Institutes of nal nitric oxide synthase-like immunoreactivity (nNOS-LI) is Health Pain Center (UCSF), C-522 Box 0452, University of California at San Francisco, San Francisco, CA 94143-0452. expressed in small- and medium-diameter dorsal root ganglion Copyright © 1998 Society for Neuroscience 0270-6474/98/187008-07$05.00/0 (DRG) neurons in rat and monkey (Zhang et al., 1993; Qian et Aley et al. • NO Signaling in Pain and Nociceptor Sensitization J. Neurosci., September 1, 1998, 18(17):7008–7014 7009

Figure 1. Reduction of mechanical no- ciceptive threshold (hyperalgesia) pro- duced by PGE2 15 min after injection (PGE2) (100 ng; n ϭ 12), L-NMA (1 ␮ ϭ g; n 6) plus PGE2 (L-NMA/PGE2) ϭ Ͻ (n 12; p 0.05), PGE2 5 min after injection [PGE2(5Ј)](n ϭ 6), L-NMA 5 Ј min after PGE2 (PGE2/L-NMA5 post) (n ϭ 6; p Ͻ 0.05), D-NMA (10 ␮g) plus ϭ PGE2 (D-NMA/PGE2)(n 6), ODQ (1 ␮ ϭ g) plus PGE2 (ODQ/PGE2)(n 6), and L-NMA (n ϭ 6) on mechanical paw- withdrawal threshold in rats. In this and subsequent figures, *p Ͻ 0.05. Higher values indicate greater hyperalgesia. The data for one behavioral experi- mental group, PGE2 , is repeated in more than one figure for ease of comparison.

al., 1996); (2) NADPH–diaphorase activity (Vanhatalao et al., PGE2 , and we investigated mechanisms underlying the induction 1996) and nNOS-LI (Zhang et al., 1993; Majewski et al., 1995) are of hyperalgesia by NO, which have not been well delineated. colocalized in some DRG neurons with substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI); and (3) MATERIALS AND METHODS NOS-LI is coexpressed in some DRG neurons with CGRP-LI or Animals. Experiments were performed on male Sprague Dawley rats SP-LI (Zhang et al., 1993) and is greatly reduced in DRG (200–250 gm; Bantin-Kingman, Fremont, CA). Animals were housed in neurons in neonatal rats treated with capsaicin (Ren and Ruda, groups of two under a 12 hr light/dark cycle. Food and water were 1995). NO is considered to play a role in the induction of noci- available ad libitum. All behavioral testing was done between 10:00 A.M. and 4:00 P.M. Experiments were performed with the approval of the ception, because NOS-LI in lumbar DRG neurons is increased by Institutional Animal Care Committee of the University of California at noxious irritation of the bladder (Vizzard et al., 1996), by noxious San Francisco. stimulation with resiniferatoxin (Farkas-Szallasi et al., 1995; Viz- Behavioral testing. The nociceptive flexion reflex was quantified with a zard et al., 1995), and by induction of neuropathic models of pain Basil analgesymeter (Stoelting, Chicago, IL), which applies a linearly using sciatic nerve transection (Fiallos-Estrada et al., 1993; Zhang increasing mechanical force to the dorsum of the rat’s hindpaw. Before the experiments, rats were exposed to the paw-withdrawal testing pro- et al., 1993; Beesley, 1995) or by ligation of lumbar dorsal roots cedure for 3 hr (1 hr/d for 3 d). On the day of the experiment, rats were (Choi et al., 1996) or nerves (Steel et al., 1994). NO also contrib- exposed to the same procedure for 1 hr, and the baseline threshold was utes to withdrawal hyperalgesia in rats made tolerant to opioid- determined as the mean of the six readings before the administration of Ϯ induced peripheral antinociception (Aley and Levine, 1997a,b). the test agent (Aley and Levine, 1997a,b). The mean SEM baseline G threshold before treatments for the rats used in these experiments was Furthermore, the NOS inhibitor N -methyl-L-arginine (L-NMA) 108.0 Ϯ 0.4 gm (n ϭ 260). Mechanical threshold was redetermined at suppresses activity in lumbar dorsal roots originating from a three time points (15, 20, and 25 min) after administration of a hyper- sciatic neuroma (Wiesenfeld-Hallin et al., 1993). L-NMA also algesic agent. The mean of these three readings was considered to be the reduces thermal hyperalgesia produced by chronic constriction paw-withdrawal threshold because of hyperalgesic agent administration, injury of the sciatic nerve or hindpaw inflammation (Moore et al., and this value was used to calculate the percentage change from the baseline threshold for each paw. To determine the timing of onset of 1993; Thomas et al., 1996; Lawand et al., 1997). Clearly, there is action of the hyperalgesic agents, the mechanical threshold was also abundant evidence that NO plays a role in nociceptive signaling. measured at 1 min intervals for 5 min after their administration, whereas When studying NO, it is important to evaluate cGMP, because the time course was determined by measuring the mechanical threshold NO stimulates guanylyl cyclase, and many of the cellular effects of at 30–60 min intervals for 2–4 hr. Drug administration. The following drugs used in this study were NO are the result of NO-induced increases in the level of cGMP obtained from Sigma (St. Louis, MO): PGE2 , L-arginine (L-Arg), G (Jaffrey and Snyder, 1995). NO-donating compounds stimulate D-arginine (D-Arg), L-NMA, N -methyl-D-arginine (D-NMA), 8-bromo- marked elevation of cGMP levels in cultured rat DRG neurons cAMP, and 3-morpholino-sydnonimine (SIN-1). Forskolin, 2-p-(2- (Dymshitz and Vasko, 1994), and activation of NOS by the algesic carboxyethyl)phenethylamino-5Ј-N-ethylcarboxamido adenosine HCl Ϯ agent bradykinin increases cGMP in rat DRG neurons (Harvey (CGS21680), and ( )-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydro- naphthalene HBr (8-OH-DPAT) were obtained from Research Bio- and Burgess, 1996). chemicals (Natick, MA). WIPTIDE was obtained from Peninsula Lab- Because interactions between inflammatory mediators have oratories (Belmont CA), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- been reported and because there are interactions at the second one (ODQ) and protein kinase A catalytic subunit (PKACS) were messenger level, it is important to evaluate whether NO contrib- obtained from Calbiochem (La Jolla, CA). The selection of the drug doses used in this study was based on dose–response curves determined utes to the prototype hyperalgesia and sensitization produced by during this and previous studies (Aley and Levine, 1997a,b). The stock PGE . In this study, therefore, we tested such a contribution by ␮ ␮ 2 solution of PGE2 (1 g/2.5 l) was prepared in 10% ethanol, and further NO to hyperalgesia produced by the inflammatory mediator dilutions were made in saline; the final concentration of ethanol was 7010 J. Neurosci., September 1, 1998, 18(17):7008–7014 Aley et al. • NO Signaling in Pain and Nociceptor Sensitization

Figure 2. L-NMA reduces the PGE2-induced poten- tiation of TTX-R INa. TTX-R INa was monitored by test pulses given every 20 sec. Voltage of the test pulse (Ϫ20 to Ϫ5 mV) was selected for each neuron to give an approximately half-maximal current am- plitude. One hundred micromolar D-NMA or L-NMA was included in the bath for 15 min before ␮ exposure to 1 M PGE2. PGE2 typically causes a potentiation of TTX-R INa in approximately half of cells tested (Gold et al., 1996a,b). Experiments were alternated between using the active and inactive en- antiomers of the NOS inhibitor, and data from all neurons were used, including those in which the cur- rent was not affected by PGE2. Peak current ampli- tudes were normalized to mean of the baseline measurements.

Figure 3. Reduction of the paw- withdrawal threshold by the A2 adeno- sine agonist CGS21680 after injection (1 ␮g; n ϭ 6), L-NMA plus CGS21680 (L-NMA/CGS)(n ϭ 6; p Ͻ 0.05), 5HT1A agonist 8-OH-DPAT after injec- tion (1 ␮g; n ϭ 6), and L-NMA plus 8-OH-DPAT (L-NMA/8-OH)(n ϭ 6; p Ͻ 0.05) on mechanical paw- withdrawal threshold in rats.

Յ1%. 8-bromo-cAMP, L-NMA, D-NMA, PKACS, and WIPTIDE U/ml each penicillin and streptomycin (UCSF Cell Culture Facility). were dissolved in saline. ODQ was dissolved in DMSO and diluted with Ganglia were dissected free, desheathed in cold culture medium, and saline (final concentration of DMSO, 10%). CGS21680 and 8-OH-DPAT then incubated for 2 hr at 37°C in culture medium with 0.125% collage- were dissolved in deionized water. All drugs administered intradermally nase. After an additional 10 min digestion in 0.25% trypsin, cells were were in a volume of 2.5 ␮l/paw. For test agents with low cell membrane mechanically dispersed by trituration with a fire-polished Pasteur pipette. permeability (i.e., WIPTIDE and PKACS), 2 ␮l of distilled water was Cells were plated onto glass coverslips coated with laminin (Life Tech- coinjected first, in the same syringe as the test agent to produce hypo- nologies) and poly-DL-ornithine (Sigma) and were maintained in culture osmotic shock and thus facilitate cell permeability (Taiwo and Levine, medium with nerve growth factor (Life Technologies) at 37°C under 3% 1989; Khasar et al., 1995). When drug combinations were used, they were CO2. Neurons were used within 24 hr of plating before appreciable administered from the same syringe in such a way that the drug men- outgrowth of neurites at a time when small diameter (20–30 ␮m) neu- tioned first reached the intradermal site first. Such combinations of ronal cell bodies express properties of nociceptors (Gold et al., 1996b). agents were separated in the syringe by a small air bubble to prevent their Drugs were added via the bath, which continuously perfused the record- mixing in the syringe. Whenever an inhibitor was included, it was ing chamber at 1–2 ml/min. Experiments were performed at room injected first. temperature (21–24°C). Cell culture and in vitro electrophysiology. Primary cultures of adult rat Whole-cell patch-clamp recordings were performed on small diameter lumbar DRG neurons were prepared as described previously (Gold et al., (Ͻ30 ␮m) neurons in 1-d-old cultures of dissociated DRGs from adult 1996b). Culture medium consisted of minimal essential medium [Uni- rats, using an Axopatch 200B amplifier with pClamp6 acquisition and versity of California at San Francisco (UCSF) Cell Culture Facility] with stimulation programs (Axon Instruments, Foster City, CA). Data were 10% fetal bovine serum (Life Technologies, Gaithersburg, MD) and 1000 low-pass-filtered at 5 kHz and acquired at a sampling rate of 10 kHz. Aley et al. • NO Signaling in Pain and Nociceptor Sensitization J. Neurosci., September 1, 1998, 18(17):7008–7014 7011

Figure 4. Reduction of the paw- withdrawal threshold by forskolin (10 ␮g) 5 min after injection (Forsk)(n ϭ 8), L-NMA plus forskolin (L-NMA/ Forsk)(n ϭ 6; p Ͻ 0.05), WIPTIDE plus forskolin (WIPTIDE/Forsk)(n ϭ 10; p Ͻ 0.05), 8-bromo-cAMP after injection (8brcAMP)(1␮g; n ϭ 12), L-NMA plus 8-bromo-cAMP (L-NMA/ 8brcAMP)(n ϭ 8; p Ͻ 0.05), WIP- TIDE plus 8-bromo-cAMP (WIP- TIDE/8brcAMP)(n ϭ 6; p Ͻ 0.05), PK ACS (15 U; n ϭ 12), WIPTIDE plus PKACS (WIPTIDE/PK ACS)(n ϭ 6; p Ͻ 0.05), and L-NMA plus PKACS (L-NMA/PK ACS)(n ϭ 6; not statisti- cally significant) on mechanical paw- withdrawal threshold in rats.

⍀ ␮ Voltage-clamp experiments were performed with 2–5 M electrodes of 1 M PGE2. The mean PGE2-induced increase in the size of filled with (in mM): CsCl 140, NaCl 10, CaCl2 0.1, MgCl2 2, EGTA 11, peak TTX-R I elicited by a depolarizing voltage step was HEPES 10, MgATP 2, and LiATP 1, pH adjusted to 7.2 with Tris base. Na significantly smaller in the cells treated with L-NMA than in those Bath consisted of (in mM): NaCl 35, tetraethylammonium chloride 30, treated with D-NMA ( p Ͻ 0.05) (Fig. 2). choline chloride 65, CaCl2 0.5, MgCl2 5, HEPES 10, and glucose, pH adjusted to 7.4 with NaOH, and osmolality adjusted to 325 mOsm with sucrose. Tetrodotoxin (50 nM) was added to the bath. Capacitance and L-NMA blocks hyperalgesia induced by CGS21680 series resistance was compensated (Ͼ80%), and leak subtraction was and 8-OH-DPAT performed with a P/4 protocol. After obtaining a current–voltage rela- We tested whether NOS activity is required to produce hyperal- tionship for TTX-R INa , a 25 msec depolarizing test pulse was applied every 20 sec to monitor the size of the current during the experiment. A gesia induced by other peripherally acting hyperalgesic agents. As voltage that produced approximately half of the maximal current acti- shown previously (Taiwo and Levine, 1989, 1990, 1991; Taiwo et vation was used for the test pulse, because the greatest increase in al., 1992), the intradermal injection of CGS21680 (A2 adenosine TTX-R I produced by PGE is seen at this part of the current–voltage ␮ Na 2 receptor agonist; 1 g) and 8-OH-DPAT (5HT1A serotonergic relationship. Experimental and control neurons were studied alternately agonist; 1 ␮g) produced mechanical hyperalgesia (Fig. 3). Injec- on the same day for each comparison. ␮ Statistical analysis. Data are presented as mean Ϯ SEM; statistical tion of L-NMA (1 g) with CGS21680 and 8-OH-DPAT signifi- significance was determined by ANOVA followed by Scheffe’s post hoc cantly attenuated the resulting hyperalgesia ( p Ͻ 0.05) (Fig. 3), Ͻ test; and p 0.05 was considered statistically significant. similar to its effect on PGE2-induced hyperalgesia.

RESULTS L-NMA blocks hyperalgesia induced by 8-bromo-cAMP and forskolin but not by PKACS L-NMA, but not ODQ, blocks PGE2 hyperalgesia To determine whether the contribution of NO to hyperalgesia is We first determined whether NO contributes to PGE2-induced hyperalgesia and whether guanylyl cyclase is involved (Fig. 1). attributable to interaction of NO with the cAMP second messen- Injection of L-NMA (1 ␮g), a competitive NOS inhibitor, preced- ger system and at what level in the cAMP second messenger pathway NO is required, we evaluated the effect of L-NMA on the ing PGE2 in the same syringe significantly attenuated PGE2- induced hyperalgesia to mechanical stimuli ( p Ͻ 0.05). Injection hyperalgesia produced by different components of the pathway. of L-NMA (1 ␮g) 5 min after PGE , when hyperalgesia is already Five minutes after the intradermal injection of 8-bromo-cAMP 2 ␮ ␮ well established (Ouseph et al., 1995), significantly reversed hy- (10 g), forskolin (10 g), or PKACS (the catalytic subunit of peralgesia. L-NMA alone had no effect on mechanical nociceptive PKA; 15 U), near-maximal mechanical hyperalgesia was present. ␮ threshold. The inactive stereoisomer of L-NMA, D-NMA (10 Injection of L-NMA (1 g) before 8-bromo-cAMP and forskolin, ␮ but not PKACS, resulted in reduced hyperalgesia (Fig. 4). Injec- g), was without effect on PGE2-induced hyperalgesia. Injection ␮ tion of WIPTIDE inhibited PKACS hyperalgesia, as it did of the guanylyl cyclase inhibitor ODQ (1 g) before PGE2 had no effect. 8-bromo-cAMP and forskolin hyperalgesia, which indicates that the isolated catalytic subunit of PKA appears to produce hyper- L-NMA inhibits PGE2-induced potentiation of TTX-R Ina algesia through the same catalytic action as PKA. in vitro

PGE2 sensitizes cultured small diameter DRG neurons and po- Administration of NO donor or precursor induces hyperalgesia tentiates TTX-R INa (England et al., 1996; Gold et al., 1996a). We tested whether endogenous NO derived from the primary affer- As shown in Figure 5, A and D, intradermal injection of the NOS ␮ ent is necessary for potentiation of TTX-R INa by PGE2. One substrate L-Arg (10 ng to 10 g) or the NO donor SIN-1 (10 ng to hundred micromolar L-NMA (n ϭ 15) or D-NMA (n ϭ 14) was 40 ␮g) caused a dose-dependent decrease in the paw-withdrawal added to the extracellular bath for 15 min before the application threshold. This hyperalgesia was inhibited by the guanylyl cyclase 7012 J. Neurosci., September 1, 1998, 18(17):7008–7014 Aley et al. • NO Signaling in Pain and Nociceptor Sensitization

Figure 5. A, Dose–response curve for L-Arg-induced hyperalgesia (n ϭ 6). B, Latency to onset of L-Arg-induced (10 ␮g) mechanical hyperalgesia (n ϭ 6). C, Time course of L-Arg-induced mechanical hyperalgesia (n ϭ 6). D, Dose–response curve of SIN-1-induced hyperalgesia (n ϭ 6). E, Latency to onset of SIN-1-induced hyperalgesia (10 ␮g; n ϭ 6). F, Time course of SIN-1-induced hyperalgesia (n ϭ 8). inhibitor ODQ but was unaffected by the PKA inhibitor WIP- observed when endogenous NOS was not inhibited (i.e., in the TIDE (Fig. 6A). This hyperalgesic action of NO contrasts with absence of L-NMA). The 100 ng dose of SIN-1 that facilitated the guanylyl cyclase-independent mechanism by which NO facil- PGE2-induced hyperalgesia was insufficient to induce hyperalge- itates PGE2-induced hyperalgesia (Fig. 6B). In control experi- sia by itself (Fig. 5D). To further preclude the possibility that ments, D-Arg did not induce hyperalgesia (Fig. 6A). NO-induced (cGMP-dependent) hyperalgesia played a role in Exogenous sources of NO can reconstitute the this reconstitution experiment, the guanylyl cyclase inhibitor ODQ was coinjected with PGE2 , L-NMA, and SIN-1. facilitatory effect of endogenous NO on PGE2-induced hyperalgesia We tested whether NO from exogenous sources can mimic the DISCUSSION ability of endogenous NO to facilitate PGE2-induced hyperalge- In this study, we found that NO, but not cGMP, contributes to sia. To inhibit endogenous NO production, L-NMA was coin- initiation and maintenance of hyperalgesia and sensitization pro- jected into the paw with PGE2 , which reduced the PGE2-induced duced by the inflammatory mediator PGE2. We also found that decrease in paw-withdrawal threshold by ϳ62% (Fig. 1). As the independent hyperalgesia produced by NO depends on shown in Figure 6B, coinjection of the NO donor compound cGMP and may require a higher concentration than that for

SIN-1 with L-NMA and PGE2 restored the PGE2-induced de- facilitation of PGE2 hyperalgesia and sensitization. The reduction crease in paw-withdrawal threshold to a value similar to that in PGE2-induced potentiation of TTX-R INa in L-NMA treated Aley et al. • NO Signaling in Pain and Nociceptor Sensitization J. Neurosci., September 1, 1998, 18(17):7008–7014 7013

Figure 6. A, Change in mechanical paw withdrawal after injection of L-Arg (L-Arg) (10 ␮g; n ϭ 12), ODQ plus L-Arg (ODQ/L-Arg)(n ϭ 6),WIPTIDE ϭ ϭ ϭ ␮ ϭ plus L-Arg (WIPTIDE/L-Arg)(n 6), L-NMA plus L-Arg (L-NMA/L-Arg)(n 6), D-NMA/L-Arg (n 6), D-Arg (D-Arg) (10 g; n 6), PGE2 (PGE2) ϭ ϭ ϭ ␮ ϭ (n 12), WIPTIDE plus PGE2 (WIPTIDE/PGE2)(n 12), ODQ plus PGE2 (ODQ/PGE2)(n 8), SIN-1 (10 g; n 6), ODQ plus SIN-1 (ODQ/SIN) ϭ ϭ ϭ (n 8), and WIPTIDE plus SIN-1 (WIPTIDE/SIN)(n 6). B, Change in mechanical paw-withdrawal threshold after injection of PGE2 (n 12), ϭ ϭ L-NMA plus PGE2 (L-NMA/PGE2)(n 6), SIN-1 (SIN) (100 ng; n 6), and L-NMA plus ODQ plus PGE2 plus SIN-1 (L-NMA/ODQ/PGE2/SIN) (100 ng; n ϭ 12).

cells parallels the effect of L-NMA on PGE2-induced hyperalge- messenger pathway and not on the cAMP pathway (Fig. 6B). We sia, suggesting that both phenomena depend on NO for full hypothesize that a relatively low level of NO can facilitate cAMP- expression and that cells other than the one being recorded from dependent hyperalgesia induced by PGE2 and other inflamma- are not required for these effects of PGE2 or NO. Although many tory mediators, whereas stimulated increases in NO to higher effects of NO have been reported to be mediated by guanylyl levels might be required to induce the cGMP-dependent hyper- cyclase activity (Jaffrey and Snyder, 1995), we observed that the algesia. Supporting this hypothesis, when endogenous NO pro- guanylyl cyclase inhibitor ODQ had no effect on PGE2-induced duction was blocked, the level of NO donor required to facilitate hyperalgesia, suggesting that facilitation of PGE -induced hyper- 2 PGE2-induced hyperalgesia was at least an order of magnitude algesia by NO and the other mechanisms of PGE2-induced hy- lower than the level required for NO to induce hyperalgesia by peralgesia do not involve NO signaling through guanylyl cyclase. itself. However, it is not possible to know whether exogenous NO Additional experiments attempted to determine where NO has ready access to the necessary sites. Also, we cannot determine might act to facilitate PGE2 hyperalgesia. Evidence suggests that from our data whether basal levels of constitutively synthesized initiation of PGE2-induced hyperalgesia is attributable to action NO are sufficient to facilitate PGE2-induced hyperalgesia or of PGE2 at an E-type prostaglandin receptor on the primary whether PGE stimulates synthesis of NO (to a level lower than afferent nociceptor terminal, activation of a stimulatory 2 that required to induce cGMP-dependent hyperalgesia). How- G-protein which then activates adenylyl cyclase, followed by an ever, it appears that constitutive synthesis of NO does not affect increase in the level of cAMP and activation of PKA (Taiwo and baseline nociceptive threshold, because inhibition of NOS does Levine, 1989, 1991; Khasar et al., 1995). We observed that hy- not alter paw-withdrawal threshold. Finally, although our in vitro peralgesia produced by 8-bromo-cAMP (which activates PKA) patch-clamp data imply that the NO that facilitates PGE -induced and forskolin (which activates adenylyl cyclase) was attenuated by 2 hyperalgesia is synthesized in DRG neurons and acts on DRG L-NMA in a manner similar to the effect of L-NMA on PGE2 hyperalgesia, but that in contrast, hyperalgesia produced by in- neurons, our data do not determine the site of action for cGMP- jection of PKACS (to mimic activity of endogenous PKA) was dependent NO-induced hyperalgesia. Clearly, further study is not affected by L-NMA. These results suggest that NO might be needed to determine concentration and site requirements for the two distinct effects of NO that we have evaluated. required for activation of PKA after administration of PGE2.Of note, others have shown that NO can modulate activity of kinases NO is generated in significant concentrations at sites of inflam- (Gopalakrishna et al., 1993; Burgstahler and Nathanson, 1995; mation in which multiple hyperalgesic inflammatory mediators, Studer et al., 1996; Minamino et al., 1997). The fact that L-NMA such as PGE2 , adenosine, or serotonin, are also produced. Al- inhibited hyperalgesia induced by two other direct-acting hyper- though the evidence for a role of NO in clinical pain is limited, algesic agents (CGS21680, an A2 adenosine receptor agonist, and NO may facilitate the hyperalgesia induced by those mediators 8-OH-DPAT, a 5HT1A serotonin receptor agonist) suggests that using the cAMP second messenger pathway and may also have an dependence on NO is a general feature of hyperalgesia induced independent cGMP-dependent hyperalgesic effect. If both of via the cAMP second messenger system. these contributions are clinically significant, different therapies Additional experiments revealed that there was an independent for these two distinct mechanisms of NO may be needed for NO-induced hyperalgesia that depended on the cGMP second successful pharmacological treatment of inflammatory pain. 7014 J. Neurosci., September 1, 1998, 18(17):7008–7014 Aley et al. • NO Signaling in Pain and Nociceptor Sensitization

REFERENCES cellular pathway through activation of protein kinase C. Circulation Aley KO, Levine JD (1997a) Different mechanisms mediate develop- 96:1586–1592. ment and expression of tolerance and dependence for peripheral mu- Moncada S, Ferreira SH, Vane JR (1975) Inhibition of prostaglandin opioid antinociception in rat. J Neurosci 17:8018–8023. biosynthesis as the mechanism of analgesia of aspirin-like drugs in the Aley KO, Levine JD (1997b) Dissociation of tolerance and dependence dog knee joint. Eur J Pharmacol 31:250–260. for opioid peripheral antinociception in rats. J Neurosci 17:3907–3912. Moore PK, Wallace P, Gaffen Z, Hart SL, Babbedge RC (1993) Char- Beesley JE (1995) Histochemical methods for detecting nitric oxide syn- acterization of the novel nitric oxide synthase inhibitor 7-nitro indazole thase. Histochem J 27:757–769. and related indazoles: antinociceptive and cardiovascular effects. Br J Burgstahler AD, Nathanson MH (1995) NO modulates the apicolateral Pharmacol 110:219–224. cytoskeleton of isolated hepatocytes by a PKC-dependent, cGMP- Moulton PJ (1996) Inflammatory joint disease: the role of cytokines, independent mechanism. Am J Physiol 269:G789–G799. cyclooxygenases and reactive oxygen species. Br J Biomed Sci Choi Y, Raja SN, Moore LC, Tobin JR (1996) Neuropathic pain in rats 53:317–324. is associated with altered nitric oxide synthase activity in neural tissue. Ouseph AK, Khasar SG, Levine JD (1995) Multiple second messenger J Neurol Sci 138:14–20. systems act sequentially to mediate rolipram-induced prolongation of Collier HO, Schneider C (1972) Nociceptive response to prostaglandins prostaglandin E2-induced mechanical hyperalgesia in the rat. Neuro- and analgesic actions of aspirin and morphine. Nat New Biol 236:141–143. science 64:769–776. Davis KD, Meyer RA, Campbell JN (1993) Chemosensitivity and sen- Qian Y, Chao DS, Santillano DR, Cornwell TL, Nairn AC, Greengard P, sitization of nociceptive afferents that innervate the hairy skin of Lincoln TM, Bredt DS (1996) cGMP-dependent protein kinase in monkey. J Neurophysiol 69:1071–1081. dorsal root ganglion: relationship with nitric oxide synthase and noci- Dymshitz J, Vasko MR (1994) Nitric oxide and cyclic guanosine 3Ј,5Ј- ceptive neurons. J Neurosci 16:3130–3138. monophosphate do not alter neuropeptide release from rat sensory Ren K, Ruda MA (1995) Nitric oxide synthase-containing neurons in neurons grown in culture. Neuroscience 62:1279–1286. sensory ganglia of the rat are susceptible to capsaicin-induced cytotox- England S, Bevan S, Docherty RJ (1996) PGE2 modulates the icity. Neuroscience 65:505–511. tetrodotoxin-resistant sodium current in neonatal rat dorsal root gan- Robbins RA, Grisham MB (1997) Nitric oxide. Int J Biochem Cell Biol glion neurones via the cyclic AMP-protein kinase A cascade. J Physiol 29:857–860. (Lond) 495:429–440. Rueff A, Dray A (1993) Sensitization of peripheral afferent fibers in the Farkas-Szallasi T, Lundberg JM, Wiesenfeld HZ, Hokfelt T, Szallasi A in vitro neonatal rat spinal cord-tail by bradykinin and prostaglandins. (1995) Increased levels of GMAP, VIP and nitric oxide synthase, and Neuroscience 54:527–535. their mRNAs, in lumbar dorsal root ganglia of the rat following Schaible HG, Schmidt RF (1988) Excitation and sensitization of fine systemic resiniferatoxin treatment. NeuroReport 6:2230–2234. articular afferents from cat’s knee joint by prostaglandin E2. J Physiol Ferreira SH, Nakamura M, Castro MSA (1978) The hyperalgesic effects (Lond) 403:91–104. of prostacyclin and prostaglandin E2. Prostaglandins 16:31–37. Steel JH, Terenghi G, Chung JM, Na HS, Carlton SM, Polak JM (1994) Fiallos-Estrada CE, Kummer W, Mayer B, Bravo R, Zimmerman M, Increased nitric oxide synthase immunoreactivity in rat dorsal root Herdegen T (1993) Long-lasting increase of nitric oxide synthase im- ganglia in a neuropathic pain model. Neurosci Lett 169:81–84. munoreactivity, NADPH-diaphorase reaction and c-JUN co- Studer RK, DeRubertis FR, Craven PA (1996) Nitric oxide suppresses expression in rat dorsal root ganglion neurons following sciatic nerve increases in mesangial cell protein kinase C, transforming growth factor transection. Neurosci Lett 150:169–173. beta, and fibronectin synthesis induced by thromboxane. J Am Soc Gold MS, Reichling DB, Shuster MJ, Levine JD (1996a) Hyperalgesic Nephrol 7:999–1005. ϩ agents increase a tetrodotoxin-resistant Na current in nociceptors. Taiwo YO, Levine JD (1989) Prostaglandin effects after elimination of Proc Natl Acad Sci USA 93:1108–1112. indirect hyperalgesic mechanisms in the skin of the rat. Brain Res Gold MS, Dastmalchi S, Levine JD (1996b) Co-expression of nociceptor 492:397–399. properties in dorsal root ganglion neurons from the adult rat in vitro. Taiwo YO, Levine JD (1990) Direct cutaneous hyperalgesia induced by Neuroscience 71:265–275. adenosine. Neuroscience 38:757–762. Gopalakrishna R, Chen ZH, Gundimeda U (1993) Nitric oxide and Taiwo YO, Levine JD (1991) Further confirmation of the role of adenyl nitric oxide-generating agents induce a reversible inactivation of pro- cyclase and of cAMP-dependent protein kinase in primary afferent tein kinase C activity and phorbol ester binding. J Biol Chem hyperalgesia. Neuroscience 44:131–135. 268:27180–27185. Taiwo YO, Levine JD (1992) Serotonin is a directly acting hyperalgesic Harvey JS, Burgess GM (1996) Cyclic GMP regulates activation of phos- agent in the rat. Neuroscience 48:485–490. phoinositidase C by bradykinin in sensory neurons. Biochem J 539–544. Taiwo YO, Heller PH, Levine JD (1992) Mediation of serotonin hyperal- Holthusen H, Arndt JO (1994) Nitric oxide evokes pain in humans on gesia by the cAMP second messenger system. Neuroscience 48:479–483. intracutaneous injection. Neurosci Lett 165:71–74. Thomas DA, Ren K, Besse D, Ruda MA, Dubner R (1996) Application Holthusen H, Arndt JO (1995) Nitric oxide evokes pain at nociceptors of of nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester, on the paravascular tissue and veins in humans. J Physiol (Lond) 487:253–258. injured nerve attenuates neuropathy-induced thermal hyperalgesia in Jaffrey SR, Snyder SH (1995) Nitric oxide: a neural messenger. Annu rats. Neurosci Lett 210:124–126. Rev Cell Dev Biol 11:417–440. Vanhatalo S, Klinge E, Sjostrand NO, Soinila S (1996) Nitric oxide- Khasar SG, Wang JF, Taiwo YO, Heller PH, Green PG, Levine JD synthesizing neurons originating at several different levels innervate rat (1995) Mu-opioid agonist enhancement of prostaglandin-induced hy- penis. Neuroscience 75:891–899. peralgesia in the rat: a G-protein beta gamma subunit-mediated effect? Vizzard MA, Erdman SL, deGroat GW (1995) Increased expression of Neuroscience 67:189–195. neuronal nitric oxide synthase in dorsal root ganglion neurons after Lawand NB, Willis WD, Westlund KN (1997) Blockade of joint inflam- systemic capsaicin administration. Neuroscience 67:1–5. mation and secondary hyperalgesia by L-NAME, a nitric oxide syn- Vizzard MA, Erdman SL, deGroat GW (1996) Increased expression of thase inhibitor. NeuroReport 8:895–899. neuronal nitric oxide synthase in bladder afferent pathways following Majewski M, Sienkiewicz W, Kaleczyc J, Mayer B, Czaja K, Lakomy M chronic bladder irritation. J Comp Neurol 370:191–202. (1995) The distribution and co-localization of immunoreactivity to Wallace JL, Chin BC (1997) Inflammatory mediators in gastrointestinal nitric oxide synthase, vasoactive intestinal polypeptide and substance P defense and injury. Proc Soc Exp Biol Med 214:192–203. within nerve fibres supplying bovine and porcine female genital organs. Wiesenfeld-Hallin Z, Hao JX, Xu XJ, Hokfelt T (1993) Nitric oxide Cell Tissue Res 281:445–464. mediates ongoing discharges in dorsal root ganglion cells after periph- Martin HA, Basbaum AI, Kwiat GC, Goetzl EJ, Levine JD (1987) eral nerve injury. J Neurophysiol 70:2350–2353. Leukotriene and prostaglandin sensitization of cutaneous high- Zhang X, Verge V, Wiesenfeld-Hallin Z, Ju G, Bredt D, Synder SH, threshold C-mechanonociceptors in the rat. Neuroscience 22:651–659. Hokfelt T (1993) Nitric oxide synthase-like immunoreactivity in lum- Minamino T, Kitakaze M, Node K, Funaya H, Hori M (1997) Inhibition bar dorsal root ganglia and spinal cord of rat and monkey and effect of of nitric oxide synthesis increases adenosine production via an extra- peripheral axotomy. J Comp Neurol 335:563–575.